Certain types of cellular cysteine proteases may play an important role in modulating the activity of G-protein coupled receptors. The activity of this class of enzymes is closely regulated by cytoplasmic and nuclear inhibitors. The interaction of these inhibitors with the target proteases is, in part, mediated by strong hydrophobic interactions. The effects of ethyl alcohol exposure on cysteine protease activity has been studied in cell culture utilizing a PC12 cell line. Activity of these proteases appears to be strongly affected by alcohol exposure. We have previously established that exposure of PC12 cells to ethyl alcohol for 96 hours results in a decrease in calcium-stimulated protease activity. We are working towards defining a mechanism for this alcohol-induced inhibition of calcium-activated protease activity. We have preliminary evidence that calpain may be redistributed in PC12 cells as a result of ethanol exposure, and that this redistribution alters the ability of detergents to extract calpain protein from cells. We have also found that while levels of calpastatin, the endogenous inhibitor of calpain, are not significantly altered after ethanol exposure, that there may be differences in its ability to inhibit calpain based on post-translational modifications. One of the possibilities is that the phosphorylation of serine residues in the protein is altered after ethanol exposure, and we are in the process of determining the extent and significance of these alterations in modulating the inhibitory activity of calpastatin after ethanol exposure. - toxicology, neurosciences, molecular genetics